Follistatin-related protein (FSRP) is a recently discovered glycoprotein whose gene structure and activin binding properties are highly homologous to the activin binding protein follistatin (FS). FSRP was originally cloned as an overexpressed protein in a B-cell leukemia and is expressed in numerous cancer cell lines, suggesting it may be involved with, or be a useful marker for a wide array of tumors. Unlike FS, however, FSRP is most highly expressed in the placenta and testis (FS is highest in the ovary and kidney), indicating that FSRP has unique functions in these tissues. Immunocytochemical studies have demonstrated that FSRP is highly concentrated in the nucleus of all cell lines and primary cells tested, but is secreted only by cells with the highest FSRP expression levels. These observations suggest that regulation of FSRP intracellular trafficking is complex and unique, and further, that FSRP may have nuclear activities distinct from strictly non-nuclear FS. Overexpression of FSRP in transgenic mice disrupts follicular development, resulting in female infertility. Thus, the broad goal of this proposal is to determine the biological functions of FSRP in normal and pathophysiological circumstances, as well as to elucidate the biochemical features of this protein which govern its unique biology and distribution. Transgenic and knockout mice, along with several in vitro bioassays will be utilized to identify the normal and pathophysiological actions of FSRP (Specific Aim 1). Intracellular trafficking, regulation of biosynthesis, and nuclear functions of FSRP will be examined in HeLa and human granulosa cells using pulse chase labeling, immunoprecipitation, and affinity chromatography (Specific Aim 2). The binding affinity and ligand specificity, as well as functional domains of FSRP responsible for these activities and FSRP's nuclear localization will be examined using site directed mutagenesis and domain swapping with FS (Specific Aim 3). The results of the proposed research program will define the role(s) of FSRP in normal physiology, determine the mechanism whereby FSRP overexpression disrupts folliculogenesis, and define the novel regulatory mechanisms that results in nuclear localization and activity of a protein that is also secreted.